TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation
Edited by Michael Karin, University of California San Diego, La Jolla, CA, and approved August 4, 2021 (received for review May 10, 2020)
Significance
The NLRP3 inflammasome is an innate sensor activated by signals released from pathogens or injured tissues. Activation of the NLRP3 inflammasome can be beneficial during infection and vaccination. Nonetheless, when NLRP3 activity is uncontrolled and chronic it becomes detrimental and contributes to inflammation-driven pathology in several diseases. Licensing mechanisms must exist that prevent unwanted NLRP3 inflammasome responses. Here, we characterize one such mechanism. We describe that TBK1 and IKKε, two closely related kinases activated upon TLR signaling, act as a novel OFF switch for the NLRP3 pathway. Using pharmacological and genetic approaches, we show that TBK1 and IKKε together limit the responses downstream of the NLRP3 inflammasome activation and work against the PP2A phosphatase ON switch to balance NLRP3 activity.
Abstract
NACHT, LRR, and PYD domains–containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a “parking brake” for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.
Data Availability
All study data are included in the article and/or supporting information.
Acknowledgments
We thank Professor Kevin Maloy for the NLRP3 KO mice and Professor Sebastian Joyce for helpful discussion during the project design and for editing the manuscript. We thank Professor David Brough from the University of Manchester for the iBMDM-ASC cell line. We thank Dr. Val Millar and Dr. Daniel Ebner from the Target Discovery Institute at University of Oxford for providing access to high-content imaging instrumentation. J.S.B. is supported by a Kennedy Trust KTRR start-up fellowship (KENN 15 16 06) and a Medical Research Council New Investigator Grant (MR/S000623/1). F.A.F. and S.D. are supported by Kennedy Trust KTPS Studentships. The work carried out in the Oxford Drug Discovery Institute was supported by Alzheimer’s Research UK Grant ARUK-2015DDI-OX.
Supporting Information
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Copyright © 2021 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
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All study data are included in the article and/or supporting information.
Submission history
Accepted: August 4, 2021
Published online: September 13, 2021
Published in issue: September 21, 2021
Keywords
Acknowledgments
We thank Professor Kevin Maloy for the NLRP3 KO mice and Professor Sebastian Joyce for helpful discussion during the project design and for editing the manuscript. We thank Professor David Brough from the University of Manchester for the iBMDM-ASC cell line. We thank Dr. Val Millar and Dr. Daniel Ebner from the Target Discovery Institute at University of Oxford for providing access to high-content imaging instrumentation. J.S.B. is supported by a Kennedy Trust KTRR start-up fellowship (KENN 15 16 06) and a Medical Research Council New Investigator Grant (MR/S000623/1). F.A.F. and S.D. are supported by Kennedy Trust KTPS Studentships. The work carried out in the Oxford Drug Discovery Institute was supported by Alzheimer’s Research UK Grant ARUK-2015DDI-OX.
Notes
This article is a PNAS Direct Submission.
Authors
Competing Interests
Competing interest statement: M.O. and Mount Sinai School of Medicine have filed a patent on the PP2A activating compound used in SI Appendix, Fig. S3.
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TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation, Proc. Natl. Acad. Sci. U.S.A.
118 (38) e2009309118,
https://doi.org/10.1073/pnas.2009309118
(2021).
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